Luminescence is a phenomenon in which energy is specifically channeled to a molecule to produce an excited state. Return to a lower energy state is accompanied by release of a photon. Luminescence includes fluorescence, phosphorescence, chemiluminescence and bioluminescence. Bioluminescence is the process by which living organisms emit light that is visible to other organisms. Where the luminescence is bioluminescence, creation of the excited state derives from an enzyme catalyzed reaction.
During the past twenty years, high-sensitivity biochemical assays used in research and in medicine have increasingly employed luminescence and fluorescence rather than radioisotopes. This change has been driven partly by the increasing expense of radioisotope disposal and partly by the need to find more rapid and convenient assay methods. More recently, the need to perform biochemical assays in situ in living cells and whole animals has driven researchers toward protein-based luminescence and fluorescence.
Since the cloning of a luciferase from the firefly, luciferase genes have become essential components of biological research. They are used ubiquitously as reporter genes in cell culture experiments, and their use as reporters has been extended into the context of small animal imaging. Recently, it has been proposed that the luciferase protein itself could be conjugated to other proteins such as antibodies or growth factors, and these bioluminescently labeled ligands could then be used for imaging of receptor targets in small animals. The advantage of using a bioluminescent entity to label a protein over similar fluorescent or radioactive approaches is that in the context of small animal imaging the bioluminescent approach has the potential to be more sensitive.
The beetle luciferases (e.g., firefly), however, are not optimal for employment as bioluminescent tags. These luciferases are not particularly small (˜62 kDa) and are dependent on ATP, molecular oxygen, and magnesium for activity. The dependence on ATP especially would hinder the application of beetle luciferases as bioluminescent tags in vivo, since serum ATP concentrations are generally below 10 nM.
Luciferases that use coelenterazine as their substrate are more appropriate for application as bioluminescent tags, as these enzymes are not ATP dependent and in general require only molecular oxygen in addition to coelenterazine for luminescence. From this group of proteins, the luciferase from Renilla reniformis (RLuc1) is the best characterized, in addition to being of a size (36 kDa) more appropriate for use as a tag.
The limiting factor for use of RLuc as a bioluminescent tag is its limited stability under in vivo conditions. A single point mutation of RLuc (C124A) that increases the enzyme's stability several fold has been reported, however even this level of stability is insufficient for the tagging of large proteins (e.g., antibodies) that require time scales on the order of days to sufficiently distribute.
An additional limitation to the use of any of the known coelenterazine utilizing luciferases is that the spectral peaks of these luciferases lie in the blue region of the visible spectrum. For in vitro assays such as cell culture transfection studies, the wavelength of light that a luciferase yields is usually of little consequence. For in vivo assays such as small animal imaging studies, the wavelength is important because biological tissues are less attenuating to the red and near-infrared portions of the optical spectrum. In the case of Renilla Luciferase the spectral peak is at 482 nm, with only about 3-4% of the photons of wavelengths above 600 nm. For luciferase at depths greater than superficial depths, the majority of the photons that actually make it out of the animal are these few above 600 nm wavelength photons.
As such, there is a continued need in the art for the development of luciferases that exhibit improved properties. The present disclosure addresses this and other needs.